Spore formation in Myxococcus xanthus is tied to cytoskeleton functions and polysaccharide spore coat deposition

Abstract

Summary Myxococcus xanthus is a Gram-negative bacterium that differentiates into environmentally resistant spores. Spore differentiation involves septation-independent remodelling of the rod-shaped vegetative cell into a spherical spore and deposition of a thick and compact spore coat outside of the outer membrane. Our analyses suggest that spore coat polysaccharides are exported to the cell surface by the Exo outer membrane polysaccharide export/polysaccharide co-polymerase 2a (OPX/PCP-2a) machinery. Conversion of the capsule-like polysaccharide layer into a compact spore coat layer requires the Nfs proteins which likely form a complex in the cell envelope. Mutants in either nfs, exo, or two other genetic loci encoding homologs of polysaccharide synthesis enzymes, fail to complete morphogenesis from rods to spherical spores and instead produce a transient state of deformed cell morphology before reversion into typical rods. We additionally provide evidence that the cell cytoskeletal protein, MreB, plays an important role in rod to spore morphogenesis and for spore outgrowth. These studies provide evidence that this novel gram-negative differentiation process is tied to cytoskeleton functions and polysaccharide spore coat deposition.

Synthesis and Assembly of a Novel Glycan Layer in Myxococcus xanthus Spores

“…Reversion is preceded by a transient stage of cell fragility and severe shape deformations (branching and formation of spiral cells) that are reminiscent of phenotypes observed in certain Escherichia coli mutants defective in peptidoglycan synthesis (10). Electron microscopy analyses revealed that an exoC mutant lacks an obvious spore coat, whereas a ⌬nfsA-H mutant produces a highly amorphous unstructured spore coat reminiscent of capsular material (5). Together, these observations suggest that assembly of a rigid, stress-bearing spore coat prior to removal of the periplasmic peptidoglycan is an essential step of spore differentiation.…”

“…In contrast, the M. xanthus spore coat material is primarily carbohydrate-rich and must be deposited on the outside of the outer membrane in a process that is directed from within the sporulating cell. Interestingly, in M. xanthus, the peptidoglycan layer appears to be degraded during spore differentiation, and the spore coat layer likely replaces its function (5,8).…”

“…Our previous work identified two genetic loci, exoA-H and nfsA-H, important for production and assembly of the spore coat, respectively (5,9). Disruption of either (or both) of these loci results in a phenotype in which cells initiate shape re-arrangement from rod to sphere but subsequently revert to rodshaped cells.…”

“…Some of the Exo proteins share homology with polysaccharide secretion machinery (5,11), and the Nfs proteins share homology with proteins (Glt) that may power the M. xanthus gliding motility machinery (12). It has been demonstrated that both the Glt and Nfs machinery appear to be involved in protonmotive force-dependent possessive transport processes, but the consequence and details of this mechanism remain undiscovered (13).…”

“…The composition and assembly of the spore cell envelope varies considerably between different spore formers, and many details of the respective assembly process have not yet been determined (3)(4)(5)(6). In the case of B. subtilis, the spore coat is composed of an altered and thickened peptidoglycan sacculus (termed the spore cortex) surrounded by a protein-rich coat (4); some spores additionally contain a mucous layer apparently composed of soluble peptidoglycan fragments (7).…”